A novel molecule Me6TREN promotes angiogenesis via enhancing endothelial progenitor cell mobilization and recruitment

Abstract

Critical limb ischaemia is the most severe clinical manifestation of peripheral arterial disease. The circulating endothelial progenitor cells (EPCs) play important roles in angiogenesis and ischemic tissue repair. The increase of circulating EPC numbers by using mobilization agents is critical for obtaining a better therapeutic outcome in patients with ischemic disease. Here, we firstly report a novel small molecule, Me6TREN (Me6), can efficiently mobilize EPCs into the blood circulation. Single injection of Me6 induced a long-lasting increase in circulating Flk-1(+) Sca-1(+) EPC numbers. In a mouse hind limb ischemia (HLI) model, local intramuscular transplantation of these Me6-mobilized cells accelerated the blood flow restoration in the ischemic muscles. More importantly, systemic administration of Me6 notably increased the capillary density, arteriole density and regenerative muscle weight in the ischemic tissue of HLI. Mechanistically, we found Me6 reduced stromal cell-derived factor-1α level in bone marrow by up-regulation of matrix metallopeptidase-9 expression, which allowed the dissemination of EPCs into peripheral blood. These data indicate that Me6 may represent a potentially useful therapy for ischemic disease via enhancing autologous EPC recruitment and promote angiogenesis.

Figure 1. Time and dose-response effects of Me6 on mobilization of EPCs into mouse PB.

(A) Time-response effects of Me6 (5 mg/kg) on mobilization of EPCs into PB. (B) Dose-response effects of Me6 on mobilization of EPCs into PB. PB were collected at 12 h post injection of Me6 and analyzed for the percentage of Flk-1⁺Sca-1⁺ cells by flow cytometry (n = 9 mice; *p < 0.05, **p < 0.01). (C–D) Representative FACS plots and mean percentages of Sca-1⁺ Flk-1⁺ cells on PBMNCs and spleen cells were shown. Mouse PB and spleen cells were collected at 12 h post injection of Me6 at 5 mg/kg or PBS. (n = 9 mice; **p < 0.01). (E) EPC-CFUs emerged from cultures of Me6 or vehicle-mobilized PBMNCs and identified by BS-lectin 1⁺ Dil-LDL⁺ cell colony units. The number of EPC colonies was counted after 12-day culture. (n = 6 mice; **p < 0.01; scale bar = 100 μm).

Figure 2. Local transplantation of Me6-mobilized PB enhances blood flow restoration in hind limb ischemia model.

(A, B) Flow cytometry analysis and quantitation of Flk-1⁺Sca-1⁺ cells on PBMNCs 12 h post injection of Me6 or 1 h post injection of AMD3100 at 5 mg/kg (n = 9 mice; **p < 0.01). (C, D) LDPI showing recovery of blood flow after surgery and expressed as the ratio of perfusion in ischemic limbs to normal limbs (n = 6 mice; AMD3100 vs. vehicle control, Me6 vs. vehicle control, *p < 0.05, **p < 0.01). Mice with HLI were locally injected with 2 mL of PB-derived MNCs from vehicle control, Me6 or AMD3100 treated mice.

Figure 3. Me6 stimulated the recovery of blood flow and increased capillary density after HLI.

(A, B) Flow cytometry analysis and quantitation of Flk-1⁺Sca-1⁺ cells on PBMNCs at 3, 7, 14 days post injection of Me6 or AMD3100 in HLI mice (n = 5 mice; *p < 0.05, **p < 0.01). (C) Representative LDPI showed recovery of blood flow after HLI surgery and injection with different agent. (D) The ratio of the ischemic (left) to normal (right) limb blood flow was used for quantitative analysis (n = 15 mice; AMD3100 vs. vehicle control, *p < 0.05; Me6 vs. vehicle control, **p < 0.01; AMD3100 vs. Me6, ^#p < 0.05). (E) Representative fluorescent microscopy images of capillaries in gastrocnemius muscle sections (BS-lectin 1⁺, green; CD31⁺, red) at day 14 (scale bar = 50 μm). Arrows indicate BS-lectin 1 and CD31 double-positive capillaries. (F) Overall the BS-lectin 1⁺CD31⁺ capillary density (n = 6 mice; *p < 0.05; ***p < 0.001) in the ischemic area.

Figure 4. Effect of Me6 on the angiogenic cell apoptosis and proliferation in ischemic limb.

(A, B) Representative microphotographs of the section of ischemic muscles stained immunochemically for TUNEL. Quantitative analysis of TUNEL⁺ apoptosis cells in ischemic hindlimb muscles at day 14 (n = 6 mice; *p < 0.05, **p < 0.01; scale bar = 100 μm). (C, D) Representative images of BrdU-DNA incorporation in BS-lectin 1⁺ endothelial cells in ischemic muscles at day 14. Quantitative analysis of BrdU⁺BS-lectin 1⁺ proliferative capillary endothelial cells (n = 6 mice in A–D; *p < 0.05, **p < 0.01; scale bar = 100 μm). (E, F) Masson's Trichrome staining (collagen stains as blue color) of ischemic muscle at day 14 (n = 6 mice; *p < 0.05, **p < 0.01; scale bar = 100 μm). (G) Tissue preservation was expressed as the ratio of muscle weight in ischemic limbs to normal limbs. The mice were killed on day 50. The wet muscular tissue of the lower limbs was isolated and weighed (n = 6 mice; *p < 0.05, ***p < 0.001).

Figure 5. Me6 enhanced the recruitment of BM-derived EPCs and angiogenesis in a HLI model.

(A) Experimental protocol. 5 × 10⁶ whole bone marrow cells from GFP-transgenic mice were transplanted into irradiated mice. Flow cytometry revealed that 85–90% of peripheral leukocytes from recipient mice were GFP⁺ at six weeks after BM reconstitution. Then these mice were performed with HLI operation and different agent injection one time per week. (B–C) Representative LDPI and mean blood flow perfusion ratio of mice with GFP⁺ BM cells replacement were analyzed after HLI surgery and injection with different agent (n = 10 mice; AMD3100 vs. vehicle control, *p < 0.05, **p < 0.01; Me6 vs. vehicle control, **p < 0.01; AMD3100 vs. Me6, ^#p < 0.05, ^##p < 0.01). (D, E) Capillaries were identified by CD31 and BS-lectin 1 staining and quantitatively expressed as a capillary number per muscle fiber on day 28. Quantification of BS-lectin 1⁺CD31⁺ capillary density (n = 6 mice; *p<0.05, **p<0.01; scale bar = 100 μm). (F) The rest of the mice in each group were killed on day 50. A histogram expressed as the ratio of muscle weight in ischemic limbs to normal limbs (n = 4 mice; *p < 0.05, **p < 0.01).

Figure 6. Histological analysis of GFP⁺ cells in ischemic tissue after HLI.

(A, B) Immunofluorescent double staining was used to analyze the GFP⁺BS-lectin 1⁺ cells in ischemic areas of the different groups and quantification of GFP⁺BS-lectin 1⁺ cells density (μm²/mm²) at day 28. (C, D) Immunofluorescent double staining was used to analyze the GFP⁺a-SMA⁺ cells in ischemic areas of the different groups and quantification of GFP⁺α-SMA⁺ vessel density (μm²/mm²) at day 28 (n = 5 mice in A–D; *p < 0.05, **p < 0.01; scale bar = 100 μm).

Figure 7. Me6 reduced SDF-1α level in the BM by up-regulation of MMP-9 expression.

(A) Fold change in the levels of SDF-1α protein in murine plasma and BM supernatants at 0–72 h post injection of Me6; the amount of SDF-1α was detected by ELISA. (B) The percentage of CXCR4⁺ BMMNCs was measured by flow cytometry at 0 h or 12 h post Me6 injection. (C) Fold change in the levels of total MMP-9 in murine BM supernatants at 0–72 h upon administration of Me6; the amount of MMP-9 was detected by ELISA. (D) The expression of MMP-9 mRNA was up-regulated in BMMNCs derived from mice with Me6 injection. Mice were sacrificed at 0, 6, 12, 24, 48 and 72 h after Me6 injection and BMMNCs were isolated for Q-PCR analysis (n = 6 mice in A–D; *p < 0.05, **p < 0.01 vs. 0 h). (E, F) Me6 treatment up-regulated the expression of MMP-9 mRNA in mouse BMMNCs and HUVECs. Mouse BMMNCs and HUVECs were cultured with Me6 for 0, 12, 24, 48 and 72 h. RNA was extracted at different time point for Q-PCR analysis. (n = 6; *p < 0.05, **p < 0.01 vs. 0 h). (G) Me6 failed to increase the percentage of circulating Flk-1⁺Sca-1⁺ cells in MMP-9^-/- mice. MMP-9^-/- mice (KO) or wild-type (WT) mice were subcutaneously injected with 5 mg/kg Me6 or vehicle. PB were collected at 12 h post injection and were analyzed for the percentage of Sca-1⁺Flk-1⁺ cells by flow cytometry (n = 4 mice; *p < 0.05 vs. WT mice with vehicle treatment).